Project Summary/Abstract Sympathetic overdrive, which results from unresolved cardiac stress, is a major driver of heart failure (HF) and has been shown to induce insulin resistance (IR) in cardiomyocytes.. The molecular mechanisms by which this takes place, however, are largely understudied. Our data show that prolonged isoproterenol (iso) treatment prevents insulin (ins)-induced increases in mitochondrial reserve respiratory capacity (RRC), a parameter that we have shown to positively correlate with ATP production and cardiomyocyte survival during energetic stress. Further, our data suggest that these effects may be mediated by an inhibition of ins-induced pyruvate dehydrogenase (PDH) activity, as we observe increased PDH kinase 1 (Pdk1) and decreased PDH phosphatase 1 (Pdp1) and ins-induced Pdp2 expression, under these conditions. Such modulations result in increased PDH phosphorylation and, therefore, decreased activity and glucose oxidation.. PDH is also a critical regulator of bioenergetics, as we have shown that PDK1 overexpression eliminates RRC, whereas PDK inhibition increases RRC, improving cardiomyocyte tolerance to stress. Lastly, our data also show that prolonged iso treatment prevents ins-induced phosphorylation and nuclear export/exclusion of the transcription factor, forkhead box protein O1 (FOXO1), resulting in its nuclear occupancy. Taken together, our data suggest that PDH and FOXO1 are novel candidates for mediating iso-induced IR in the heart. Further, as phosphorylation has been shown to regulate FOXO1 activity and localization, we propose that iso treatment increases FOXO1 phosphorylation at ins-independent sites, increasing its nuclear occupancy, and differentially regulating the expression of genes involved in IR. Thus, our central hypothesis is that prolonged ?-adrenergic stimulation induces IR in cardiomyocytes at the level of bioenergetics, decreasing their capacity for ATP production and, therefore, survivability during energetic stress, consequently accelerating the progression of HF. To test this hypothesis, our specific aims are (1) to examine the roles of PDH and FOXO1 in catecholamine (CA)-induced IR and HF at the level of mitochondrial bioenergetics and cellular survival, both in vitro and in vivo and (2) to investigate the mechanisms by which FOXO1 mediates the effects of CA-induced IR. To achieve aim 1, we will utilize gain- and loss-of-function approaches to examine the roles of PDK1, PDP1/2, and FOXO1 in iso-induced IR in vitro and in vivo, as well as the roles of PDK1 and PDP1/2 in a murine model of cardiac hypertrophy and failure. For aim 2, we will utilize in vitro approaches to validate Pdk1 as a FOXO1 transcriptional target, as well as evaluate the role FOXO1 phosphorylation in iso-induced IR. Overall, this proposal examines the molecular mechanisms of prolonged ?-adrenergic-induced IR in the heart, information that may allow us to inhibit this process, thereby, boosting myocardial energetics and cellular survival and, consequently, slowing the progression of HF.